Abstract Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a key effector function of therapeutic monoclonal antibodies (mAb) in use for treatment of established cancers. mAbs communicate with NK cells via the low-affinity Fc? receptor IIIa (Fc?RIIIa). mAbs, bound to targets on cancer cells, promote crosslinking of Fc?RIIIa and activation of NK cells leading to both cytotoxicity and cytokine responses. Polymorphisms in Fc?RIIIa determine the avidity for mAb with the higher affinity variant correlating with more effective ADCC and better clinical outcomes. As a result much of the development efforts of next generation therapeutic mAbs has centered on engineering mAbs with improved Fc?RIIIa engagement. Interleukin-2 (IL-2) promotes the activation, proliferation and cytotoxicity of NK cells. Recombinant IL-2 is approved for use in humans and has proven anti-cancer effects but short half-life and substantial toxicities have limited its use. Fusion of IL-2 to mAbs to produce `immunocytokines' has several advantages over co- administration of IL-2 + mAb such as reducing the amount of IL-2 administered, thereby reducing toxicities, directing IL-2 to NK cells that express Fc?RIIIa, the very subset of NK cells that initiates ADCC, and localizing IL-2 to the site of disease where NK cell activation may be most relevant to anti-tumor responses. We have developed novel modified Fc regions from human IgG1 consisting of open-hinge tandem arrays of the HCH2 region (`HCH2 polymers') that possess enhanced binding to low-affinity Fc? receptors (Fc?R). In preliminary studies we developed anti-Her2/Neu antibodies based on our HCH2 polymers and demonstrated their enhanced capacity to trigger ADCC against tumor cells. We examined the role of exogenously added IL-2 to potentiate ADCC from standard mAb (Herceptin) or from mAbs with enhanced Fc?R binding. We observed that mAbs with superior Fc?R binding also had the greatest benefit from IL-2 co- administration. Thus mAbs that have been optimized for NK cell activation via Fc?R may be ideal candidates for further modification via fusion to IL-2. One goal of the proposed studies is use our anti-Her2/Neu mAbs that are already optimized for Fc?R binding to create IL2-mAb fusions and to determine if the IL2-mAb fusions constitute a superior anti-cancer therapeutic than the unmodified antibodies. A second aspect of the proposed studies is to determine if non-targeted IL-2-optimized Fc fusions will function as an NK adjuvant for increasing ADCC responses when co-administered with anti-cancer mAbs. In Aim 1 studies we will produce IL-2 fusions to targeted (IL2-mAb) and non-targeted (IL2-HSA3) forms of the HCH2 polymers. In Aim 2 we will determine the impact of IL-2 fusion proteins on cytotoxicity, phenotype, and proliferative response of isolated NK cell subsets in vitro. We will also determine whether the IL-2-mAb fusions augment ADCC against Her2 expressing cell lines. In Aim 3 we will evaluate the antitumor effects of IL-2-mAb and IL-2-HSA3 fusions in vivo using SKOV3 xenografts in SCID mice and in an immunocompetent humanized immune system mouse model (HSC-NSG). This combination of Aims is designed to deliver sufficient proof-of-principle for advancing the HCH2 polymer-IL2 fusion proteins towards clinical development.